Cytoprotective Proteins Research Articles

Cryo-EM structure of histidyl-tRNA synthetase-like domain reveals activating crossed helices at the core of GCN2.

GCN2 is a conserved receptor kinase activating the integrated stress response (ISR) in eukaryotic cells. The ISR kinases detect accumulation of stress molecules and reprogram translation from basal tasks to preferred production of cytoprotective proteins. GCN2 stands out evolutionarily among all protein kinases due to the presence of a histidyl-tRNA synthetase-like (HRSL) domain, which arises only in GCN2 and is located next to the kinase domain (KD). How HRSL contributes to GCN2 signaling remains unknown. Here, we report a 3.2 Å cryo-EM structure of HRSL from thermotolerant yeast Kluyveromyces marxianus. This structure shows a constitutive symmetrical homodimer featuring a compact helical-bundle structure at the junction between HRSL and KDs, in the core of the receptor. Mutagenesis demonstrates that this junction structure activates GCN2 and indicates that our cryo-EM structure captures the active signaling state of HRSL. Based on these results, we put forward a GCN2 regulation mechanism, where HRSL drives the formation of activated kinase dimers. The remaining domains of GCN2 have the opposite role and in the absence of stress they help keep GCN2 basally inactive. This auto-inhibitory activity is relieved upon stress ligand binding. We propose that the opposing action of HRSL and additional GCN2 domains thus yields a regulated ISR receptor.

Abstract 4125992: Eicosapentaenoic Acid (EPA) Increases Expression of Nrf2-mediated Antioxidant Response Element and Heme Oxygenase-1 in Cytokine-Activated Endothelial Cells

Background: Endothelial cell (EC) nuclear factor erythroid 2-related factor (Nrf2) translocates to the nucleus during inflammation to mediate transcription of genes in the antioxidant response elements (ARE). An ARE regulates mRNA encoding HMOX-1, thioredoxin (TXN), and NAD(P)H quinone oxidoreductase-1 (NQO1), among others. Parkinson disease protein 7 (PARK7) and p62 work independently to prevent Keap1-mediated degradation of Nrf2 during oxidative stress. Eicosapentaenoic acid (EPA) administered as icosapent ethyl reduced CV events (REDUCE-IT). We tested the effects of EPA on ARE protein expression in ECs from multiple tissues during cytokine challenge. Methods: Human brain, pulmonary and vascular ECs were treated with IL-6 (12 ng/mL) for 2 hours and then incubated with EPA (40 µM) for 24 h. Global proteomic analysis performed by LC-MS measured relative protein expression. Significant (p<0.05) changes between treatment groups >1-fold were analyzed by differential enrichment analysis of proteomics data (DEP) and included in gene set enrichment analyses (GSEA). Significantly modulated pathways within the Gene Ontology (GO) database were identified as those with an adjusted-p value <0.05. Results: Proteomic analysis revealed that EPA significantly modulated ≥600 proteins in ECs distinct from IL-6 challenge alone. In EC, EPA significantly modulated the “cellular response to oxidative stress” pathway (GO: 0034599) relative to IL-6 alone. Within this GO pathway, EPA increased levels of catalase (1.2-fold, 1.1-fold, 1.1-fold) and mitochondrial glutathione reductase (1.1-fold, 1.1-fold, 1.1-fold) in brain, pulmonary, and vascular ECs, respectively. We also observed a consistent increase in brain, pulmonary, and vascular ECs of p62 (1.3-fold, 1.2-fold, and 1.2-fold, respectively), and ARE members HMOX-1 (1.5-fold, 1.7-fold, and 2.1-fold, respectively), and NQO1 (1.1-fold, 1.2-fold, and 1.3-fold, respectively). TXN was increased in pulmonary and vascular ECs (1.1-fold and 1.2-fold, respectively) with EPA. Mitochondrial TXN (also called TXN2) increased in brain ECs with EPA 1.1-fold. EPA also significantly increased levels of PARK7 1.1-fold. Conclusions: During inflammation, EPA enhanced expression of cytoprotective proteins in the ARE, notably HMOX-1, along with its regulators, in ECs from multiple tissues. Augmented endogenous antioxidant pathways may contribute to EPA’s clinical benefits.

Heat shock protein HSPA8 impedes hemin-induced cellular-toxicity in liver

Accumulation of hemin in cells, tissues, and organs is one of the major pathological conditions linked to hemolytic diseases like malaria. Pro-oxidant hemin confers high toxicity following its accumulation. We tested the cellular toxicity of hemin on HepG2 cells by exploring modulation in various cellular characteristics. Hemin reduces the viability of HepG2 cells and brings about visible morphological changes. Hemin causes perforations on the surface of HepG2 cells observed through SEM. Hemin leads to the extracellular release of liver enzymes and reduces the wound-healing potential of HepG2 cells. Hemin leads to the fragmentation of HepG2 DNA, arrests the cell cycle progression in the S-phase and induces apoptosis in these cells. Western blot analysis revealed that hemin triggers both the extrinsic and intrinsic pathways of apoptosis in HepG2 cells. We have already shown that the cytoprotective protein HSPA8 can polymerize hemin and minimize its toxicity. Similar experiments with hemin in the presence and absence of HSPA8 showed that HSPA8 reverses all the tested toxic effects of hemin on HepG2 cells. The protection from hemin toxicity in HepG2 cells appeared to be due to the extracellular polymerization of hemin by HSPA8.

Eicosapentaenoic acid increases expression of cytoprotective proteins in the antioxidant response element (ARE) during inflammation in endothelial cells

Eicosapentaenoic acid increases expression of cytoprotective proteins in the antioxidant response element (ARE) during inflammation in endothelial cells

Pharmacologic Inhibition of EIF4A Blocks NRF2 Synthesis to Prevent Osteosarcoma Metastasis.

Effective therapies for metastatic osteosarcoma (OS) remain a critical unmet need. Targeting mRNA translation in metastatic OS offers a promising option, as selective translation drives the synthesis of cytoprotective proteins under harsh microenvironmental conditions to facilitate metastatic competence. We assessed the expression levels of eukaryotic translation factors in OS, revealing the high expression of the eukaryotic initiation factor 4A1 (EIF4A1). Using a panel of metastatic OS cell lines and patient-derived xenograft (PDX) models, EIF4A1 inhibitors were evaluated for their ability to block proliferation and reduce survival under oxidative stress, mimicking harsh conditions of the lung microenvironment. Inhibitors were also evaluated for their antimetastatic activity using the ex vivo pulmonary metastasis assay and in vivo metastasis models. Proteomics was performed to catalog which cytoprotective proteins or pathways were affected by EIF4A1 inhibition. CR-1-31B, a rocaglate-based EIF4A1 inhibitor, exhibited nanomolar cytotoxicity against all metastatic OS models tested. CR-1-31B exacerbated oxidative stress and apoptosis when OS cells were co-treated with tert-butylhydroquinone, a chemical oxidative stress inducer. CR-1-31B potently inhibited OS growth in the pulmonary metastasis assay model and in experimental and spontaneous models of OS lung metastasis. Proteomic analysis revealed that tert-butylhydroquinone-mediated upregulation of the NRF2 antioxidant factor was blocked by co-treatment with CR-1-31B. Genetic inactivation of NRF2 phenocopied the antimetastatic activity of CR-1-31B. Finally, the clinical-grade EIF4A1 phase-1-to-2 inhibitor, zotatifin, similarly blocked NRF2 synthesis and the OS metastatic phenotype. Collectively, our data reveal that pharmacologic targeting of EIF4A1 is highly effective in blocking OS metastasis by blunting the NRF2 antioxidant response.

Application of Bioinformatics and Molecular Dynamics Simulation for Designing FGF7 Mutants Stable in Urea Solution

Background: FGF7 is a potent cytoprotective and regenerative protein on the injured epithelial tissues, indicating its potential therapeutic effect in conditions such as mucositis, ulcerative colitis, and cutaneous wound formation induced by chemotherapy or radiotherapy. However, FGF7 low stability prevents its usability as a pharmaceutical product, necessitating the design and production of stable FGF7 mutants. Methods: In the current study, bioinformatics and MD simulations approaches were used for identifying potential stable mutants of FGF7. For this, the model structure of FGF7 was constructed and subjected to MAESTROweb service to identify stability conferring mutations. The identified potential mutants of FGF7 were MD simulated in an explicit denaturant condition and their stabilities were evaluated. The complexes of FGF7 mutants with FGFR2 receptor were also examined to investigate the receptor binding ability of proposed mutants. Results: Investigation of stability parameters showed that introducing A104S mutations in FGF7 may greatly improve its stability in urea solution. Calculation of binding energies indicated that A104S mutant interacts better then wild type FGF7 with the receptor. The structural stability and binding capability of A104S were comparable to that of A120C mutation, as an experimentally determined stability conferring mutation, which was used in whole process to indicate the validity of employed MD simulation for predicting the effect of mutation on FGF7 stability. Conclusion: The results of current study showed that A104S mutant of FGF7 has a potential to be evaluated further for the purpose of presenting a therapeutic agent effective in conditions such as mucositis, ulcerative colitis, and cutaneous wound formation induced by chemotherapy or radiotherapy.

SARS-CoV-2 S Protein Reduces Cytoprotective Defenses and Promotes Human Endothelial Cell Senescence.

Premature vascular aging and endothelial cell senescence are major risk factors for cardiovascular diseases and atherothrombotic disturbances, which are main complications of both acute and long COVID-19. The S protein of SARS-CoV2, which acts as the receptor binding protein for the viral infection, is able to induce endothelial cells inflammation and it has been found as an isolated element in the circulation and in human tissues reservoirs months after infection. Here, we investigated whether the S protein is able to directly induce endothelial cell senescence and deciphered some of the mechanisms involved. In primary cultures of human umbilical vein endothelial cells (HUVEC), SARS-CoV-2 S protein enhanced in a concentration-dependent manner the cellular content of senescence and DNA damage response markers (senescence-associated-β galactosidase, γH2AX), as well as growth-arrest effectors (p53, p21, p16). In parallel, the S protein reduced the availability of cytoprotective proteins, such as the anti-aging protein klotho, Nrf2 or heme oxygenase-1, and caused functional harm by impairing ex vivo endothelial-dependent vasorelaxation in murine microvessels. These effects were prevented by the pharmacological inhibition of the NLRP3 inflammasome with MCC950. Furthermore, the supplementation with either recombinant klotho or angiotensin-(1-7), equally protected against the pro-senescence, pro-inflammatory and pro-oxidant action of the S protein. Globally, this study proposes novel mechanisms of disease in the context of COVID-19 and its vascular sequelae and provides pharmacological clues in order to prevent such complications.

Modulation of the Prostate Cancer Resistance Factor Hsp27 by the Chemotherapeutic Drugs Abiraterone, Cabazitaxel, Docetaxel and Enzalutamide.

The cytoprotective heat shock protein 27 (HSP27) acts as a protein chaperone, antioxidant, and apoptosis regulator and is involved in cytoskeletal remodeling in prostate cancer. This study was designed to assess the effect of prostate cancer therapeutics on HSP27 to identify drugs that may benefit from an HSP27 inhibitor combination therapy. Cell counting was utilized to assess drug treatment efficiency. Changes in protein levels after drug treatment were assessed using western blot analysis. Abiraterone, cabazitaxel, docetaxel and enzalutamide significantly reduced cell proliferation in LNCaP and PC3 cells. Treatment with abiraterone and enzalutamide led to a significant reduction in HSP27 protein levels. In contrast, treatment with cabazitaxel and docetaxel did not change the HSP27 protein levels. Treatment with abiraterone and enzalutamide reduces HSP27 protein in an AR-independent manner and thus suppresses HSP27-correlated resistance mechanisms. However, docetaxel and cabazitaxel do not alter HSP27 protein levels, so that taxanes' efficacy may be enhanced by combining them with HSP27-inhibiting drugs.

Cytoprotective 3K3A-activated protein C and plasma: A comparison of therapeutics for the endotheliopathy of trauma.

Both healthy plasma and cytoprotective aPC (3K3A-aPC) have been shown to mitigate the endotheliopathy of trauma (EoT), but optimal therapeutics remain unknown. Our aim was therefore to determine optimal therapies to mitigate EoT by investigating the effectiveness of 3K3A-aPC with and without plasma-based resuscitation strategies. Electric cell-substrate impedance sensing (ECIS) was used to measure real-time permeability changes in endothelial cells. Cells were treated with a 2 μg/mL solution of aPC 30 minutes prior to stimulation with plasma taken from severely injured trauma patients (ISS > 15 and BD < -6) (TP). Healthy plasma, or plasma frozen within 24 hours (FP24), was added concomitantly with TP. Cells treated with thrombin and untreated cells were included in this study as control groups. A dose-dependent difference was found between the 5% and 10% plasma-treated groups when HUVECs were simultaneously stimulated with TP (μd 7.346 95%CI 4.574 to 10.12). There was no difference when compared to TP alone in the 5% (μd 5.713 95%CI -1.751 to 13.18) or 10% group (μd -1.633 95%CI -9.097 to 5.832). When 3K3A-aPC was added to plasma and TP, the 5% group showed improvement in permeability compared to TP alone (μd 10.11 95%CI 2.642 to 17.57), but there was no difference in the 10% group (μd -1.394 95%CI -8.859 to 6.070). The combination of 3K3A-aPC, plasma, and TP at both the 5% plasma (μd -28.52 95%CI-34.72 to -22.32) and 10% plasma concentrations (μd -40.02 95%CI -46.22 to -33.82) had higher inter-cellular permeability than the 3K3A-aPC pre-incubation group. Our data shows that FP24, in a post-trauma environment, pre-treatment with 3K3A-aPC can potentially mitigate the EoT to a greater degree than FP24 with or without 3K3A-aPC. Although further exploration is needed, this represents a potentially ideal and perhaps superior therapeutic treatment for the dysregulated thromboinflammation of injured patients. Prognostic/Epidemiological, Therapeutic/Care Management, Level III.

EGCG-vanadium nanomedicine with neutral pH Fenton reaction activity inhibits heat shock proteins for enhanced photothermal/chemodynamic therapy

A burgeoning interest has recently focused on the development of nanomedicine to integrate noninvasive photothermal therapy (PTT) and chemodynamic therapy (CDT) for synergistic tumor treatments, owing to PTT's amplification effect on CDT. However, challenges emerge as hyperthermia often induces an unwarranted overexpression of cytoprotective heat shock proteins (HSPs), thereby curtailing PTT efficacy. Additionally, the nearly neutral tumor intracellular pH (pHi ≈ 7.2) that handicaps the Fenton reaction poses a leading limitation to CDT. Addressing these hurdles, we introduce EVP, a nanomedicine developed through the straightforward assembly of epigallocatechin gallate (EGCG), vanadium sulfate (VOSO4), and Pluronic F-127 (PF127). EVP comprehensively downregulates overexpressed HSPs (HSP 60, 70, 90) through the collaborative action of EGCG and vanadyl (VO2+). Moreover, the tumor intracellular pH-processed Fenton-like reaction by VO2+ ensures highly efficient hydroxyl radicals (OH) production in cytosols, overcoming the stringent acidity requirement for CDT. Additionally, the hyperthermia induced by PTT augments OH production, further enhancing CDT efficacy. In vitro and in vivo experiments validate EVP's excellent biocompatibility and potent tumor inhibition, highlighting its substantial potential in tumor therapy.

Digital manufacturing techniques and the in vitro biocompatibility of acrylic-based occlusal device materials

ObjectivesMaterial chemistry and workflow variables associated with the fabrication of dental devices may affect the biocompatibility of the dental devices. The purpose of this study was to compare digital and conventional workflow procedures in the manufacturing of acrylic-based occlusal devices by assessing the cytotoxic potential of leakage products.MethodsSpecimens were manufactured by 3D printing (stereolithography and digital light processing), milling, and autopolymerization. Print specimens were also subjected to different post-curing methods. To assess biocompatibility, a human tongue epithelial cell line was exposed to material-based extracts. Cell viability was measured by MTT assay while Western blot assessed the expression level of selected cytoprotective proteins.ResultsExtracts from the Splint 2.0 material printed with DLP technology and post-cured with the Asiga Flash showed the clearest loss of cell viability. The milled and autopolymerized materials also showed a significant reduction in cell viability. However, by storing the autopolymerized material in dH2O for 12 h, no significant viability loss was observed. Increased levels of cytoprotective proteins were seen in cells exposed to extracts from the print materials and the autopolymerized material. Similarly to the effect on viability loss, storing the autopolymerized material in dH2O for 12 h reduced this effect.Conclusions/Clinical relevanceBased on the biocompatibility assessments, clinical outcomes of acrylic-based occlusal device materials may be affected by the choice of manufacturing technique and workflow procedures.

Ethanol-induced lung and cardiac right ventricular inflammation and remodeling underlie progression to pulmonary arterial hypertension.

Current research on ethanol-induced cardiovascular anomalies has focused on left ventricular (LV) function and blood pressure. To extend this area of research, we sought to determine whether ethanol-induced alterations in the structure and function of the right cardiac ventricle (RV) and pulmonary artery (PA) lead to pulmonary arterial hypertension (PAH). Two groups of male Sprague-Dawley rats received a balanced liquid diet containing 5% ethanol (w/v) or a pair-fed isocaloric liquid diet for 8 weeks. Weekly echocardiography was conducted to evaluate cardiopulmonary function, and lung and RV tissues were collected for exvivo histological and molecular studies. The ethanol-treated rats exhibited: (1) Elevated mean pulmonary arterial pressure and decreased pulmonary artery acceleration time/ejection time; (2) Pulmonary vascular remodeling comprising intrapulmonary artery medial layer thickening; and (3) RV hypertrophy along with increased RV/LV + septum, RV diameter, RV cardiomyocyte cross-sectional area, and LV mass/body weight ratio. These responses were associated with increased lung and RV pro-inflammatory markers, endothelin-1 (ET-1), TNF-α, and IL-6 levels and higher ET-1,ET-1 type A/B receptor ratio, and downregulation of the cytoprotective protein, bone morphogenetic protein receptor 2 (BMPR2), in the lungs. These findings show that moderate ethanol-induced cardiopulmonary changes underlie progression to PAH via an upregulated proinflammatory ET1-TNFα-IL6 pathway and suppression of the anti-inflammatory BMPR2.

Cytoprotective mechanism of cerebro-cognitive reserve

IntroductionConsideration of the reserve problem would be incomplete without an analysis of the cytoprotective mechanism. The predominant molecular hallmark of aging and degeneration is the accumulation of altered gene products. Moreover, several conditions, including protein, lipid, or glucose oxidation, disrupt redox homeostasis and lead to the accumulation of unfolded or misfolded proteins in the aging brain in case of AD, and other neurodegenerative diseases that have as a common denominator abnormal protein production, mitochondrial dysfunction and oxidative stress. Some authors classify aging, pathological aging, and neurodegeneration as “protein conformational diseases”.Objectivesscientific publicationsMethodsanalytical reviewResults The central nervous system has evolved a conserved unfolded protein response mechanism to cope with the accumulation of misfolded proteins. As one of the main intracellular redox systems involved in neuroprotection, the vitagene system becomes a potential neurohormetic target for novel cytoprotective interventions. Vitagens encode the cytoprotective heat shock proteins (Hsp) Hsp70 and heme oxygenase-1, as well as thioredoxin reductase and sirtuins. The cellular stress response is the ability of a cell to withstand stressful conditions, including the heat shock response. The production of heat shock proteins, including protein chaperones, is necessary for the folding and repair of damaged proteins, which promotes cell survival to avoid apoptosis.«Molecular chaperone» are proteins that function as part of an ancient defense system in our cells. They promote cell survival by sequestering damaged proteins and preventing their aggregation. Chaperone complexes are involved in the regulation of mitochondrial functions, assembly of the cytoplasmic proteolytic system of brain cells. The cellular response to stress requires the activation of survival pathways that are under the control of protective genes called vitagens. Vitagens are involved in the production of heat-shock protein molecules, glutathione, and bilirubin. They have antioxidant and anti-apoptotic activity and provide protection against oxidative stress.Conclusions Studies have shown that the heat shock response contributes to the maintenance of cellular homeostasis, the establishment of a cytoprotective state in a wide range of human diseases, including inflammation, cancer, aging, and neurodegenerative disorders. Endogenous proteins can be manipulated by food or pharmacological compounds, which represents an innovative approach to therapeutic intervention in neurodegenerative disorders, actually influencing reserve mechanisms and adaptive capacity.Disclosure of InterestNone Declared

Abstract 7200: Non-cytotoxic inhibition of nuclear encoded mitochondrial protein MAGMAS resensitize treatment resistant prostate cancer cells to taxane therapy

Abstract Metastatic prostate cancer (PCa) is the leading cause of PCa-associated deaths and one of the leading causes of cancer mortality in men. Novel therapeutics, such as Prostate Specific Membrane Antigen (PSMA) ligand therapy, are emerging as effective treatment strategies for metastatic prostate cancer. However, most men succumb to Metastatic Castration Resistant Prostate Cancer (mCRPC) due to treatment resistance. MAGMAS, a 13.8 kDa mitochondrial protein, imports nuclear-encoded mitochondrial proteins into the mitochondrial matrix. MAGMAS is overexpressed in several aggressive tumors, such as breast cancer, glioblastoma, and prostate cancer. When overexpressed, MAGMAS plays a role as a cytoprotective protein by acting as a reactive oxygen species (ROS) scavenger that maintains sufficient ROS levels to promote cell proliferation but below the threshold levels that could lead to apoptosis. This study was designed to investigate the pro-survival and therapy-resistance functions of MAGMAS in treatment-resistant PCa cells. Predictively, we observed that the docetaxel (DTX)-resistant PC3-DR and DU145-DR PCa cell lines expressed significantly higher levels of MAGMAS compared to their drug-sensitive parental cell lines. We hypothesized that inhibiting Magmas reduces the aggressive properties of highly proliferative DTX-resistant metastatic PCa cell lines and re-sensitizes them to DTX. MAGMAS knockdown in DTX-resistant PC3 and DU145 cells in the presence of increasing DTX treatments enhanced sensitivity to DTX. Consistent with these results, pharmacological inhibition of MAGMAS with the novel BT#9 inhibitor in combination with increasing DTX concentrations also enhanced sensitivity to DTX in these cell lines. Additional studies using cell migration/invasion assays and tumor-sphere formation assays are underway to further establish MAGMAS as a potential therapeutic target to attenuate the aggressive properties of chemo-resistant PCa cells. Lastly, we will investigate the diagnostic potential of MAGMAS as a biomarker for advanced PCa and BT#9 as an imaging/therapeutic-specific agent for treatment-resistant tumors. Citation Format: Alfonso Duran, Kristen Whitley, Krystal Santiago, Adil Mohammed, Carlos Casiano, Bhaskar Das, Frankis Almaguel. Non-cytotoxic inhibition of nuclear encoded mitochondrial protein MAGMAS resensitize treatment resistant prostate cancer cells to taxane therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7200.

Na+/K+-ATPase: ion pump, signal transducer, or cytoprotective protein, and novel biological functions.

Na+/K+-ATPase is a transmembrane protein that has important roles in the maintenance of electrochemical gradients across cell membranes by transporting three Na+ out of and two K+ into cells. Additionally, Na+/K+-ATPase participates in Ca2+-signaling transduction and neurotransmitter release by coordinating the ion concentration gradient across the cell membrane. Na+/K+-ATPase works synergistically with multiple ion channels in the cell membrane to form a dynamic network of ion homeostatic regulation and affects cellular communication by regulating chemical signals and the ion balance among different types of cells. Therefore, it is not surprising that Na+/K+-ATPase dysfunction has emerged as a risk factor for a variety of neurological diseases. However, published studies have so far only elucidated the important roles of Na+/K+-ATPase dysfunction in disease development, and we are lacking detailed mechanisms to clarify how Na+/K+-ATPase affects cell function. Our recent studies revealed that membrane loss of Na+/K+-ATPase is a key mechanism in many neurological disorders, particularly stroke and Parkinson's disease. Stabilization of plasma membrane Na+/K+-ATPase with an antibody is a novel strategy to treat these diseases. For this reason, Na+/K+-ATPase acts not only as a simple ion pump but also as a sensor/regulator or cytoprotective protein, participating in signal transduction such as neuronal autophagy and apoptosis, and glial cell migration. Thus, the present review attempts to summarize the novel biological functions of Na+/K+-ATPase and Na+/K+-ATPase-related pathogenesis. The potential for novel strategies to treat Na+/K+-ATPase-related brain diseases will also be discussed.

ARTICULAR CHONDROCYTES FROM DIABETIC AND NON-DIABETIC RATS EXPOSED TO NORMO- OR HYPERGLYCAEMIA RESPOND DIFFERENTIALLY TO THE COMPLEMENT SPLIT FRAGMENT ANAPHYLATOXIN C5A AND TNFA

Osteoarthritis (OA) and diabetis mellitus type 2 (DMT2) are pathogenetically linked. Complement dysregulation contributes to OA and could be involved in DMT2. The inflammatory anaphylatoxin C5a is released during complement activation. This study aims to understand the specific responses of chondrocytes isolated from diabetic and non-diabetic rats exposed to C5a and/or the proinflammatory cytokine TNFα in vitro dependent on the glucose supply. Articular chondrocytes of adult Zucker Diabetic Fatty (ZDF) rats (homozygous: fa/fa, diabetic, heterozygous: fa/+, lean controls) were exposed to 10 ng/mL TNFα and 25 ng/mL C5a alone or in combination, both, under normo- (NG, 1 g/L glucose) and hyperglycemic (HG, 4.5 g/L glucose) conditions (4 or 24 h). Chondrocyte survival, metabolic activity and gene expression of collagen type 2, suppressors of cytokine signaling (SOCS)1, −3 and anti-oxidative hemoxygenase-1 (HMOX1) were assessed. The complement regulatory protein CD46 and cell nuclei sizes were analyzed. Chondrocyte vitality remained unaffected by the treatment. Metabolic activity was impaired in chondrocytes of non-diabetic rats under HG conditions. Collagen type 2 transcription was suppressed by TNFα under HG condition in chondrocytes from nondiabetic donors and under both conditions in those of DMT2 rats (24 h)Except for DMT2 chondrocytes under HG (4 h), HMOX1 was generally induced by TNFα +/- C5a (NG, HG). C5a elevated HMOX1 only in chondrocytes of controls. The SOCS1/3 genes were increased by TNFα (NG, diabetic, non diabetic, 4 and 24 h). This could also be observed in chondrocytes of diabetic, but not of lean rats (24 h, HG). At 4 h, C5a induced SOCS1 only in non diabetic chondrocytes (NG, HG). Cytoprotective CD46 protein was suppressed by TNFα under NG condition. Nuclear volumes of chondrocyte were lower in chondrocytes from DMT2 rats compared to those from controls. The differential response suggests that chondrocytes are irreversibly compromised by DMT2.Achnowledgement: The authors are grateful for the support by the “Stiftung Edoprothetik (S 04/21)”

Expression of a stress-inducible heme oxygenase-1 in NK cells is maintained in the process of human aging.

Heme oxygenase-1 (HO-1) is a stress-inducible heat shock protein (HSP32) that exerts cytoprotective effects against oxidative stress and inflammation, and is involved in the maintenance of cellular homeostasis. This study aimed to evaluate the expression of HO-1 in natural killer (NK) cells from individuals of different age groups after stimulation with various factors, and to analyze the relationships between the concentration of this cytoprotective protein and parameters corresponding to oxidative stress and inflammation, that is, NOD-like receptor protein 3 (NLRP3), glutathione (GSH), GSH disulfide (GSSG), and interleukin 6 (IL-6). The study population comprised three age groups: young adults (age range, 19-23 years), older adults aged under 85 years (age range, 73-84 years), and older adults aged over 85 years (age range, 85-92 years). NLRP3, GSH, and GSSG concentrations were measured in serum, whereas the HO-1 concentration and IL-6 expression were studied in NK cells cultivated for 48h and stimulated with IL-2, lipopolysaccharide (LPS), or phorbol 12-myristate 13-acetate (PMA) with ionomycin. The analysis of serum NLRP3, GSH, and GSSG concentrations revealed no statistically significant differences among the studied age groups. However, some typical trends of aging were observed, such as a decrease in GSH concentration and an increase in both GSSG level, and GSSG/GSH ratio. The highest basal expression of IL-6 and lowest basal content of HO-1 were found in NK cells of adults over 85 years of age. The NK cells in this age group also showed the highest sensitivity to stimulation with the applied factors. Moreover, statistically significant negative correlations were observed between HO-1 and IL-6 expression levels in the studied NK cells. These results showed that NK cells can express HO-1 at a basal level, which was significantly increased in activated cells, even in the oldest group of adults. The reciprocal relationship between HO-1 and IL-6 expression suggests a negative feedback loop between these parameters.

Structural modification of C2-substituents on 1,4-bis(arylsulfonamido)benzene or naphthalene-N,N′-diacetic acid derivatives as potent inhibitors of the Keap1-Nrf2 protein-protein interaction

The Keap1-Nrf2-ARE signaling pathway is an attractive therapeutic target for the prevention and treatment of oxidative stress-associated diseases by activating the cellular expression of cytoprotective enzymes and proteins. Small molecule inhibitors can directly disrupt the Keap1-Nrf2 protein-protein interaction (PPI), resulting in elevated levels of Nrf2 protein and subsequent stimulation of related antioxidant responses. Previously, we found that 1,4-bis(arylsulfonamido)benzene or naphthalene-N,N′-diacetic acid derivatives with an ether type C2-substituent on the benzene or naphthalene core exhibited potent inhibitory activities with IC50's in the submicromolar or nanomolar range. We here describe a more detailed structure-activity relationship study around the C2 substituents containing various polar linkers shedding new insight on their binding interactions with the Keap1 Kelch domain. The key observation from our findings is that the substituents at the C2-position of the benzene or naphthalene scaffold impact their inhibitory potencies in biochemical assays as well as activities in cell culture. The biochemical FP and TR-FRET assays revealed that the naphthalene derivatives 17b and 18 with an additional carboxylate at the C2 were the most active inhibitors against Keap1-Nrf2 PPI. In the cell-based assay, the two compounds were shown to be potent Nrf2 activators of the transcription of the Nrf2-dependent genes, such as HMOX2, GSTM3, and NQO1.

Generation of CRISPR-Cas9 edited human induced pluripotent stem cell line carrying BAG3 V468M mutation in its BAG domain

BCL2-Associated Athanogene 3 (BAG3) gene was identified mutated in patients with dilated cardiomyopathy (DCM), an important cause of heart failure and premature death. BAG3 is a cytoprotective co-chaperonne protein involved in many cellular process with a central role in the maintenance of protostasis. We generated two human induced pluripotent stem cell lines (hiPSc), one carrying the heterozygous, the other the homozygous p.V468M mutation identified in DCM familial cases. All lines expressed pluripotent markers, had normal karyotype, and differentiated into derivatives of the three germ layers. Sudies of hiPSc derived cardiomyocytes will help to understand the role of BAG3 in DCM.

Endogenous Protein C Activation with AB002 Improves Outcomes in a Rat Model of Acute Ischemic Stroke

Background: The hemorrhagic side-effects of current thrombolytic agents limit their usefulness to a small subset of acute ischemic stroke patients. An early, effective, and hemostatically safe antithrombotic/neuroprotectant in addition to standard of care could improve outcomes. AB002 (E-WE thrombin) is an engineered recombinant thrombin analog (W215A/E217A) that activates cytoprotective and antithrombotic endogenous protein C on intravascular surfaces without any measurable procoagulant activity. In renal failure patients undergoing hemodialysis, AB002 treatment reduced dialyzer clotting without increasing markers of hemostasis impairment or coagulation (NCT03963895). Preclinical studies also showed that AB002 exhibits potent antithrombotic/thrombolytic effects in a non-human primate model of acute vascular graft thrombosis, as well as reduces infarct size and improves outcomes in an experimental acute myocardial ischemia-reperfusion model in mice. Aims: Here, we evaluated whether neuroprotective effects of AB002 can be observed following middle cerebral artery occlusion-recanalization (MCAO-R) in rats. Methods: The local animal care and use committee approved all studies. 8 to 9-week-old male rats (275-377 g) were anesthetized under isoflurane and a small laser-Doppler probe was affixed to the skull to monitor cortical perfusion and verify vascular occlusion and reperfusion. A silicone-coated 4-0 nylon monofilament was inserted into the right internal carotid artery (ICA) via the external carotid artery (ECA) until the laser-Doppler signal dropped to &amp;lt; 30 % of baseline. To evaluate the effect of AB002 on outcomes, rats (n = 4/ group) were treated with 25 µg/kg (0.25 mL/kg; i.v.) or saline placebo, administered 30 min after induction of ischemia. Since prolongation of the activated partial plasminogen time (aPTT) is a pharmacologic effect of high dose drug exposure, a 0.45 mL blood sample was collected at 20 min post treatment from the right jugular vein, processed to plasma and frozen for subsequent aPTT evaluation. At 90 min, the filament was removed, the surgical incisions were sutured closed and postoperative animals were monitored during recovery. Neurological performance scores and weight were assessed daily for 4 days. Rats were sacrificed on day 7 post-surgery and infarct size was measured using 2,3,5-triphenyltetrazolium chloride (TTC) staining. To account for the effect of edema, the infarcted area was estimated indirectly by subtracting the non-infarcted area in the ipsilateral hemisphere from the contralateral hemisphere and expressing infarct volume as a percentage of contralateral hemisphere. APTT was measured in duplicate using a KC-4 coagulometer. All analyses were performed by researchers blinded to treatment. Results: Compared to placebo, rats that received AB002 exhibited less ischemic damage, evidenced by a statistically significant reduction in infarct volume of the cortex (32.3% versus 8.9%, P &amp;lt; 0.05) and hemisphere (28.0% versus 9.8%, P &amp;lt; 0.01). In addition, rats had better neurological scores and greater weight gains during recovery following treatment with AB002, compared to placebo. Mean aPTT values were higher in AB002 treated rats compared to placebo (34 ± 2.2 s versus 21 ± 1.6 s), confirming drug exposure. Conclusions: Treatment with AB002 during ischemia reduced infarct volume and improved outcomes in the rat experimental stroke model. Considering its neuroprotective and antithrombotic properties, we propose that early treatment with AB002 may confer outcome benefit for patients experiencing acute thrombotic emergencies.